This proposal is in response to the Request for Applications (RFA) from NIAAA entitled "Alcohol-Induced Metabolic and Hepatic Injury (AIMHI)". The goal of this multiple-PI application is to examine how ethanol exposure can lead to impaired membrane trafficking events in the liver hepatocyte that results in increased fat accumulation due to altered dynamics of large fat storage organelles termed lipid droplets (LDs). During alcoholic fatty liver disease (AFLD), almost all heavy drinkers develop fatty liver, which is marked by the aberrant and significant accumulation of intrahepatocellular fatty acids in the form of LDs. The cellular processes contributing to this marked increase in the number and size of these organelles is considered a prime target for therapeutic intervention to block further progression as it is an initial stage of the injury, and thus reversible. It appears that the cyclical formation, accumulation and subsequent metabolism of LDs are dependent on an intricate trafficking process in the hepatocyte that share marked similarities with the endocytic and secretory trafficking pathways. We have found that central to LD dynamics in hepatocytes are several GTPases (dynamins and rabs in particular) that can act as molecular switches to regulate membrane traffic. In preliminary data obtained by Drs. Casey and McNiven in a recently funded "Challenge Grant" it was shown that disruption of these GTPases (by ethanol or by experimental manipulation) could dramatically increase accumulation of LDs in the liver cell. These findings support our central hypothesis that ethanol exposure leads to an impairment of the membrane trafficking machinery in the hepatocyte that attenuates LD disassembly resulting in hepatic steatosis. The two principal investigators involved in this proposed project have complementary strengths;one is an expert in alcoholic-induced liver damage (Casey), and the other in hepatocyte membrane-cytoskeleton dynamics (McNiven). We will utilize a variety of state-of-the art membrane trafficking and imaging technologies that are novel to this area of research in our investigations of how LD formation and utilization is affected by ethanol in hepatocytes. Novel and innovative biological concepts pursued in this proposal include: one, ETOH disrupts vesiculation of LDs normally used to aid in lipolysis, two, the hepatocyte endocytic machinery is utilized in this LD vesiculation process and compromised by ETOH exposure, three, ubiquitinylation of LD proteins is markedly attenuated by ETOH and aids in targeting of the LDs to the lysosome for subsequent degradation. Successful completion of these studies will provide new technologies and insights as to how ethanol affects LD dynamics in the liver, and provide information which could lead to therapeutic strategies aimed at reducing the severity of steatosis and blocking the further progression to steatohepatitis, fibrosis and cirrhosis. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to examine how alcohol exposure can lead to impaired membrane trafficking events in the liver which would eventually result in increased fat due to altered dynamics of the large fat storage organelles, lipid droplets (LDs). We hypothesize that ethanol exposure leads to hepatic steatosis as a result of attenuated LD vesiculation and subsequent trafficking to lysosomes. This leads to altered LD disassembly which then contributes to hepatic steatosis. Successful completion of these studies will provide novel insights as to how ethanol affects LD dynamics in the liver cells, and provide information which could lead to therapeutic strategies that might reduce the severity of steatosis and block the further progression to steatohepatitis, fibrosis and cirrhosis.